MPRAsnakeflow experiment QC report

Low correlation warning!

At least one (not all) Spearman correlations across replicates is low using a barcode threshold per oligo of 10! Spearman correlation of DNA is 0.95, RNA is 0.60 and DNA/RNA ratio is 0.65. Minimum allowed values are 0.85, 0.95, and 0.75.

Overall quality metrics

Table explanation
  • median rna read count: Median of RNA read count for oligos that passed filtering to determine sufficient coverage in terms of read count. Value is the median of all replicates.
  • median barcodes passing filtering: Median number of barcodes across tested sequences that passed filtering to determine if there was sufficient barcode to oligo coverage. Value is the median of all replicates.
  • pearson correlation: The correlation of log2 RNA/DNA ratios across tested sequences as a measure of replicable activity signal. Value is the median of replicate comparisons using only oligos with >= 10 barcodes.
  • fraction oligos passing: Fraction of tested sequences that passed filtering of the mappable sequences to determine if the designed library was sufficiently recovered. Value is the median of all replicates and using only oligos with >= 10 barcodes.
median barcodes passing filtering median rna read count pearson correlation fraction oligos passing
67 641 0.85 0.92

DNA over RNA counts

Plotting normalized counts of DNA vs RNA (median across replicates). Only oligos within all replicates are shown. We should see a variation within the RNA count data (along the y axis). If count data between RNA and DNA is highly correlated (e.g. follows the identity line) there is no variation between designed oligos. This is an indication that RNA is inflated with DNA and the DNA digestion before creating cDNA did not work as expected.

Oligo correlation

Oligo correlation plots of DNA, RNA and DNA/RNA ratios across replicates. First tab shows plots using (in average) 54115 oligos with a minimum number of 10 barcodes. Second tab shows all 58442 oligos that have assigned barcodes.

Condition A B #Oligos A #Oligos B #Oligos Joined DNA spearman RNA spearman Ratio spearman DNA log2 pearson RNA log2 pearson Ratio log2 pearson
A549 1 2 54794 54706 54331 0.96 0.63 0.67 0.96 0.82 0.84
A549 1 3 54794 54778 54369 0.96 0.64 0.67 0.96 0.83 0.85
A549 1 4 54794 54341 54069 0.96 0.62 0.67 0.96 0.82 0.84
A549 1 5 54794 54333 54058 0.96 0.60 0.65 0.95 0.81 0.84
A549 2 3 54706 54778 54306 0.96 0.66 0.71 0.96 0.86 0.88
A549 2 4 54706 54341 54047 0.96 0.64 0.70 0.96 0.84 0.86
A549 2 5 54706 54333 54027 0.96 0.63 0.70 0.95 0.84 0.87
A549 3 4 54778 54341 54056 0.96 0.65 0.71 0.96 0.85 0.87
A549 3 5 54778 54333 54063 0.96 0.64 0.70 0.96 0.86 0.87
A549 4 5 54341 54333 53833 0.95 0.61 0.70 0.95 0.84 0.86
Condition A B #Oligos A #Oligos B #Oligos Joined DNA spearman RNA spearman Ratio spearman DNA log2 pearson RNA log2 pearson Ratio log2 pearson
A549 1 2 58719 58727 58503 0.95 0.60 0.63 0.91 0.73 0.73
A549 1 3 58719 58717 58482 0.95 0.61 0.64 0.91 0.74 0.74
A549 1 4 58719 58651 58425 0.95 0.59 0.64 0.90 0.71 0.73
A549 1 5 58719 58639 58414 0.95 0.57 0.62 0.90 0.72 0.73
A549 2 3 58727 58717 58498 0.95 0.63 0.67 0.91 0.77 0.77
A549 2 4 58727 58651 58438 0.95 0.61 0.66 0.90 0.74 0.75
A549 2 5 58727 58639 58434 0.95 0.60 0.66 0.90 0.75 0.76
A549 3 4 58717 58651 58428 0.95 0.61 0.67 0.90 0.75 0.76
A549 3 5 58717 58639 58423 0.95 0.61 0.66 0.90 0.77 0.77
A549 4 5 58651 58639 58380 0.94 0.58 0.66 0.90 0.74 0.76

Experiment statistic

The total number of oligos in this experiment is 59308 (defined by the assignment) with 10382452 associated barcodes.

In average across replicates we see 58691 from 4913065 average barcodes in the count data and around 1166025 barcodes where not in the assignment.

condition replicate oligos dna/rna matched barcodes unknown barcodes dna/rna % matched barcodes total dna counts total rna counts avg dna counts per bc avg rna counts per bc barcode outlier removed avg dna/rna barcodes per oligo
A549 1 58719 5079064 1199761 80.89 201932147 92956866 32.16 14.80 0 86.50
A549 2 58727 5001282 1194537 80.72 199108959 100483452 32.14 16.22 0 85.16
A549 3 58717 5121282 1231303 80.62 202834242 83176133 31.93 13.09 0 87.22
A549 4 58651 4666773 1093439 81.02 188881416 77523105 32.79 13.46 0 79.57
A549 5 58639 4696924 1111087 80.87 189555867 75199143 32.64 12.95 0 80.10
Experiment Barcodes Counts Average counts Assigned barcodes Assigned counts Average assigned counts Fraction assigned barcodes Fraction assigned counts
A549_1_DNA 15667758 299222797 19.10 9523291 251677049 26.43 0.61 0.84
A549_2_DNA 15667758 299222797 19.10 9523291 251677049 26.43 0.61 0.84
A549_3_DNA 15667758 299222797 19.10 9523291 251677049 26.43 0.61 0.84
A549_4_DNA 15667758 299222797 19.10 9523291 251677049 26.43 0.61 0.84
A549_5_DNA 15667758 299222797 19.10 9523291 251677049 26.43 0.61 0.84
A549_1_RNA 7637312 95973168 12.57 5146017 79456373 15.44 0.67 0.83
A549_2_RNA 7707693 103808893 13.47 5067807 85879123 16.95 0.66 0.83
A549_3_RNA 7658028 85918859 11.22 5191145 71064019 13.69 0.68 0.83
A549_4_RNA 6935335 80063496 11.54 4726312 66159992 14.00 0.68 0.83
A549_5_RNA 6998535 77706392 11.10 4758105 64312175 13.52 0.68 0.83

Histograms barcodes per oligo, counts per barcode

Histogramm of number of barcodes per oligo and the number of counts per barcode devidied by DNA and RNA. Median is red, mean is blue.

Activity

Violin and box plots of the log2 fold change of all oligos by the assay. Grouped by labels if set, otherwise NA. First tab shows plots using (in average) 54115 oligos with a minimum number of 10 barcodes. Second tab shows all 58442 oligos that have assigned barcodes.